This study adhered to the principles of the Declaration of Helsinki (World Medical Association, 2013), Good Clinical Practice (GCP) (NMPA, National Medical Products Administration, National Health Commission of the People’s Republic of China, 2020), and the guidelines of the National Medical Products Administration (NMPA) (NMPA, The National Medical Products Administration of China, Center for Drug Evaluation, 2019) of China. The trial was registered with the number CTR20233578, conducted at the Phase I Clinical Trial Center of Zhonghui Cardiovascular Disease Hospital, Henan (Zhengzhou), from 1 December 2023, to 4 February 2024, and approved by the Institutional Ethics Committee before initiation.

This trial involved a single-center, randomized, open, single oral administration, two-formulation, four-period crossover study and featured a screening period, four treatment periods, a washout period of 14 days after each treatment period, and a follow-up period following the last treatment (Figure 1). A total of 36 healthy male and female Chinese subjects were included in the study and randomly assigned to either the K₁ group (TRTR) or K₂ group (RTRT) with equal sex ratios using SAS software (v9.4). The subjects were admitted to the Phase I Clinical Research Center the day before dosing and fasted overnight (10 h). All subjects received a single oral administration of 0.2 g of sorafenib tosylate tablets (T formulation,0.2 g, lot SRF1230602, Renhe Xidelong Pharmaceuticals Co., Ltd.) or Nexavar^®(R formulation, 0.2 g, lot: BXJX3D1, Bayer AG) on the day of dosing with 240 mL of water. The participants were not allowed to drink additional water for 1 h before or after treatment. and forbidden from walking or eating for 4 h after administration.

All participants received group and individual counseling sessions with the investigator to understand the trial process and risks, and signed informed consent forms prior to participation.The inclusion criteria are healthy male and female subjects aged 18 years and above; male subjects weighing ≥50 kg and female subjects weighing ≥45 kg with a body mass index (BMI) of 19.0–26.0 kg/m²; full understanding of the experimental procedure and risks; and no birth plan from the beginning of the experiment to 3 months after its completion.

The exclusion criteria were participation in any other clinical trial of a drug within the past 3 months, presence of any clinically relevant medical condition or history, inability to tolerate venipuncture, blood or needle sickness, blood donation or blood loss ≥400 mL within the past 3 months, abnormal and clinically significant physical examination, electrocardiogram, vital signs, laboratory tests, and hypersensitivity to the study drug. Pregnant and lactating women were excluded from this study.

Blood samples (N = 22) were collected from before drug administration to120 h after administration for each subject at the following time points: 0 h (immediately before dosing) and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 9, 10, 11, 12, 24, 48, 72, 96 and 120 h after administration. A total of 4 mL of whole blood was collected into EDTA-K₂ vacuum blood collection tubes. The blood samples were centrifuged at 3,000 g for about 5 min at 2°C–8°C within 1 hour, All plasma samples were frozen at −70°C (−60 to −90°C) within 2 h of blood collection.

A methodologically validated UPLC-MS/MS method was used to determine the plasma concentration of sorafenib. Ultra-performance liquid chromatography was performed using the LC-40D system (Shimadzu, Japan), while the mass spectrometer was the AB Sciex QTRAP 6500+ (Applied Biosystems, United States). The data acquisition software was Analyst 1.7.3 (Applied Biosystems, United States).

Plasma samples containing the drug were processed using a protein precipitation method. The internal standards were sorafenib-d3 (QCSRM, lot number: 24213; purity: 97.51%) and sorafenib toluenesulfonate (China Academy of Food and Drug Control, lot number: 420138-202201; content: 99.7%). The internal standard precipitant was Sorafenib-d3 diluted with acetonitrile at a concentration of 20.00 ng/mL. The drug-containing plasma was thawed at room temperature and then precipitated by a 10-fold dilution with the internal standard precipitant. The mixture was vortexed for 5 min, centrifuged at 15,000 rpm and 4°C for 10 min, and the supernatant was used for UPLC-MS/MS analysis. The plasma concentration range of the standard curve for this analytical method was 20–3,000 ng/mL,and the LLOQ, LQC, MQC and HQC of the substances to be measured were 20.00, 60.00, 300.0 and 2400 ng/mL, respectively.The intra-lot accuracy deviations of all QC samples ranged from −5.5% to 5.2% with a precision maximum (%CV) of 1.25%; the inter-lot accuracy deviations ranged from −5.5% to 5.9% with a %CV of 1.72%.

Chromatographic separation of the samples was performed on an ACQUITY UPLC^®BEH C18 column (1.7 μm, 50 × 2.1 mm). Gradient elution of sorafenib was performed using 2 mM ammonium formate-water (A): acetonitrile (B) as the mobile phase at a flow rate of 0.5 mL/min and column temperature of 40°C. Positive ionization mode was used to detect sorafenib in the plasma samples. The mass-to-charge ratios (m/z) of sorafenib and sorafenib-d3 were 465.2→252.2 and 468.1→255.2 respectively.

The pharmacokinetic parameters of sorafenib were calculated using a non-compartmental method. C_max and T_max are the peak concentration and time, respectively, which were obtained directly from the measured blood concentration-time (C-T) curves. AUC_0-t is the area under the blood concentration-time curve from drug administration to the last point of blood sampling, calculated using the linear trapezoidal rule. AUC_{0- ∞} is the area under the curve from drug administration to extrapolation to infinity. t_1/2 is the elimination half-life, and these parameters and the mean sorafenib blood concentration-time plot were obtained using Phoenix WinNonlin software (V 8.3).

Statistical analysis was performed using the SAS software (V 9.4). A descriptive analysis of the subjects’ baseline characteristics, such as sex, age, height, weight, and body mass index, as well as pharmacokinetic concentration data, was performed. Means, standard deviations, maximum and minimum values, medians, and first and third quartiles (Q1,Q3) were calculated for continuous variables, while frequencies and percentages were calculated for categorical variables. The main pharmacokinetic parameters (C_max, AUC_0-t, and AUC_{0- ∞} ) were log-transformed and analyzed by analysis of variance (ANOVA) (NMPA, The National Medical Products Administration of China, Center for Drug Evaluation, 2019). The sequences, drugs, and periods were considered fixed effects, while subjects were treated as random effects in the ANOVA model. A two-sided t-test was conducted to evaluate the bioequivalence of the two formulations, and a non-parametric test of crossover design (Wilcoxon test) was used to analyze the statistical significance of the differences in the time to peak between the preparations. The T formulation was considered equivalent to the R formulation if the 90% confidence interval of the log-transformed geometric mean ratio of AUC_0-t, AUC_{0- ∞} , and C_max fell within the range of 80%–125%.

Safety analyses were performed for subjects who had received at least one dose of the test drug. The safety of sorafenib tosylate tablets was assessed based on adverse event reports, vital signs (including seated blood pressure, pulse, and temperature), physical examinations, 12-lead electrocardiograms, and clinical laboratory tests (routine blood, urine, blood biochemistry, coagulation, and virologic testing). Vital signs were measured and recorded respectively within 1 hour before and 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h after dosing during each period. In addition, the subjects underwent clinical laboratory testing, physical examination, and 12-lead electrocardiography at screening and before withdrawal from the study. All post-dose adverse events were monitored throughout the trial and assessed according to the Common Terminology Criteria for the Evaluation of Adverse Events (CTCAE V 5.0). All adverse events were coded using the Preferred Terminology of the International Medical Terminology Dictionary (MedDRA V 26.0) and categorized and summarized according to system organ classification and preferred terminology.

A total of 78 healthy adult subjects were screened in this study, of which 42 failed screening and 36 (29 males and 7 females) were successfully enrolled and randomly assigned to either the K₁ (TRTR) or K₂ (RTRT) group, which had 18 participants each. All participants received the administered drug. A total of 34 subjects completed the trial, and two subjects withdrew from the study early willingly (one in the third period and the other in the fourth period). The demographic information and baseline characteristics of the K₁ and K₂ groups were group-balanced. The demographic data for all subjects is summarized in Table 1, and the flow chart of the study is shown in Figure 1.

Data from the blood concentration studies of all 36 subjects were included in the pharmacokinetic analysis. The mean blood concentration-time curves for sorafenib after oral administration of the T formulation and R formulation under fasting conditions are shown in Figure 2. The pharmacokinetic parameters for the T and R formulations are summarized in Table 2.

The mean ± SD (CV%) of the C_max values for the T and R formulations were 1,306.7 ± 806.20 (61.7%) ng/mL and 1,451.2 ± 753.73 (51.9%) ng/mL, respectively; the AUC_0-t values were 26,759.7 ± 15,437.47 (57.7%) ng·h/mL and 32,512.8 ± 18,125.10 (55.7%) ng·h/mL, respectively; the AUC_{0- ∞} values were 28,597.5 ± 15,783.78 (55.2%) ng·h/mL and 34,410.6 ± 18,933.27 (55.0%) ng·h/mL, respectively. The median T_max values for two formulations were both 4.0 h. The mean ± SD (CV%) of the λz values for the T and R formulations were 0.0 ± 0.01 (31.7%) 1/h and 0.0 ± 0.01 (28.5%) 1/h, respectively, and the t_1/2 values were 25.8 ± 11.58 (45.0%) h and 24.3 ± 9.71 (39.9%) h, respectively.The pharmacokinetic profile of sorafenib generally exhibits enterohepatic reabsorption characterized by multiple peaks, a long clearance half-life, and slow terminal elimination.

In this study, the intra-individual standard deviation (S_WR) of C_max, AUC_0-t, and AUC_{0- ∞} of the T formulation were 0.40, 0.43, 0.42, respectively, which were all ≥0.294, so the bioequivalence evaluation was carried out by the reference-scaled average bioequivalence (RSABE) approach.The GMR point estimators of C_max, AUC_0-t, and AUC_{0- ∞} were 88.97%, 81.67%, and 83.66%, respectively, and fell within the bioequivalence standard range of 80%–125%. Meanwhile, the upper limits of the one-sided 95% confidence intervals for C_max, AUC_0-t, and AUC_{0- ∞} after the natural logarithmic transformation were −0.05, −0.04, and −0.03, respectively, which were all less than 0. In addition, the differences between T_max of the T and R formulations were not statistically significant, as shown by the Wilcoxon signed-rank test (p = 0.3650 > 0.05). These results indicate that the T and R formulations are bioequivalent under fasting conditions. The results of the bioequivalence evaluation between the T and R formulations under the fasting status are shown in Table 3.

A total of 7 instances of TEAE were reported in 5 out of the 36 subjects after administering the T formulation. All 7 instances were possibly related to the study drug, resulting in a TEAE incidence rate of 13.89% for the T formulation. In contrast, participants experienced a total of 4 instances of TEAE in 4 subjects after receiving the R formulation with only 1 instance being possibly related to the study drug, and the remaining 3 instances being possibly unrelated to the T formulation, which resulted in a TEAE incidence rate of 11.11% for the R formulation. Table 4 displays the status and comparison of adverse events between the T and R formulations.